Method for detecting and quantifying a target analyte in a sample

ABSTRACT

The invention relates to a method for identifying and quantifying by mass spectrometry at least one target analyte in a sample, comprising the following steps:
         a) depositing the sample to be analyzed on a support;   b) analyzing the sample by mass spectrometry, so as to obtain the mass spectrum of the target analyte in said sample;   c) weighting a signal associated with the mass spectrum of the target analyte in said sample by a extinction coefficient (TEC) specific to the target analyte and to the sample; and   d) using the weighted signal of the target analyte to determine the quantity of target analyte in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/425,570, filedMar. 21, 2012, now U.S. Pat. No. 9,182,409, which claims the benefit ofU.S. Provisional Patent Application 61/539,681, filed Sep. 27, 2011, thedisclosures of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to a method for detecting and quantifying at leastone target molecule (target analyte) in a sample by mass spectrometry.More particularly, the invention provides a method for detecting andquantifying a target analyte directly on the surface of a sample byusing mass spectrometry imaging, in particular matrix-assisted laserdesorption/ionization (MALDI) imaging.

Generally, the invention can be applied in any field in which thequantification of an analyte in a sample is useful or necessary. Theinvention can be applied, for example, in the pharmaceutical field tostudy the distribution and the pharmacokinetics of a drug in variousbiological tissues. Additionally, the invention can be applied in thefield of agricultural chemistry, notably to evaluate the toxicity andthe degradation of an analyte such as a herbicide in plants and/or theenvironment (soil, surrounding ground water, etc.).

STATE OF THE ART

Mass spectrometry is a technique widely known and used in chemical andbiochemical analysis to detect and identify analytes of interest in asample. Molecular imaging by mass spectrometry, such as MALDI imaging,has been developed in recent years, making it possible to visualize thedistribution of analytes of interest directly on sections of biologicaltissue. MALDI imaging, by virtue of its high sensitivity, makes itpossible to simultaneously visualize the distribution of a very largenumber of different analytes directly on the surface of a sample. In thepharmaceutical field, this technology makes it possible, for example, tocompare the distribution of an analyte in various organs at variouspoints of time during treatment.

However, if quantification of the analyte thus detected at time t isdesired, it is necessary to couple this imaging technique withquantitative chemical analysis, by a traditional or instrumental method.This second quantification step can be the source of handling andinterpretation errors. Moreover, it does not enable direct correlationbetween the presence of the analyte of interest and its quantitativedistribution in the sample.

DESCRIPTION OF THE INVENTION

The invention proposes a method generally using mass spectrometry,during a single analysis, to detect and quantify a target molecule (alsoreferred to as a “target analyte”) in a sample. Preferentially, theinventive method uses mass spectrometry imaging, which enables automatedacquisition of a signal related to the mass spectrum of the targetanalyte, directly on the sample, in order to reconstruct images of thedistribution and the quantity of said target analyte in the sample.

To this end, according to the invention, an extinction coefficient (TEC)for the target analyte, specific to each target analyte in a givensample, is defined and integrated into the method. Indeed, a givenanalyte at a given concentration does not emit a signal of the sameintensity depending on the sample in which it is detected. Similarly,two different analytes at an identical concentration in a given samplehave different signal intensities. Calculation and integration of theTEC make it possible to recognize and to take into account the signalintensity variations associated with the mass spectrum of the targetanalyte in the sample to be analyzed. The TEC can then be used tonormalize the signal obtained for said analyte, so that it isrepresentative of its concentration, independently of the nature of thesample and its location in said sample. Direct quantification of theanalyte from the mass spectrometry results obtained for the targetanalyte in the analyzed sample is thus made possible.

One object of the invention thus relates to a method for identifying andquantifying by mass spectrometry at least one target analyte in asample, comprising the following steps:

-   -   a) depositing the sample to be analyzed on a support;    -   b) analyzing the sample by mass spectrometry, so as to obtain        the mass spectrum of the target analyte in said sample;    -   c) weighting a signal associated with the mass spectrum of the        target analyte in said sample by a extinction coefficient (TEC)        specific to the target analyte and to the sample; and    -   d) using the weighted signal of the target analyte to determine        the quantity of target analyte in the sample.

The inventive method can apply to any type of sample that can beanalyzed by a mass spectrometer, whether said sample is organic orinorganic. The inventive method is also applicable to any type ofsupport that can be used in mass spectrometry (slide, plate, membrane,etc.). The method is thus particularly suited to the analysis ofbiological tissues. In this case, tissue sections are prepared,typically on the order of several micrometers thick, and are depositedon a support, such as a slide, enabling their introduction into the massspectrometer. The inventive method can also be used for the analysis ofenvironmental samples, such as samples of soil, water, plants, etc.

During step a), the sample can be deposited by any known technique,i.e., manually (for example by means of a pipette), or automatically (byusing a spotting apparatus, or by spraying or sublimation, for example).The sample can be diluted or treated before deposition on the samplesupport.

Step b) of analysis of the target analyte can be performed by any massspectrometry method, notably using direct mass spectrometry (MS) ortandem mass spectrometry (MS^(n), MRM, SRM).

The experimental parameters, such as mass range and/or laser intensity,are advantageously set so as to optimize detection of the target analytein terms of intensity, sensitivity and resolution.

The mass spectra are then acquired.

For step c), various spectral characteristics can be used as the signal,notably the intensity of the peaks of the mass spectrum, thesignal-to-noise ratio (S/N), the area of the peak, etc.

An important step of the method lies in the normalization of themeasured signal. To this end, the spectral characteristic selected asthe signal for the target analyte in the sample is weighted by anextinction coefficient (TEC) specific to the analyte and to the sample.This weighting normalizes the signal and makes it dependent only on thequantity of the analyte at the origin of the signal.

The (TEC) is representative of the loss or gain in intensity of thetarget analyte's signal according to the nature of the sample and/or itslocation on the sample, compared to the signal of said analyte on aninert sample support. The (TEC) is dependent on several factors, notablythe sample's origin (animal, plant, bacterium, inorganic), the surfacetype (tissue, plant cell, metals, etc.), the chemical environment, thepresence or absence of chemical treatment of the sample, etc. In thecase of a sample of biological tissue, the extinction coefficient of theanalyte corresponds to the extinction coefficient of the tissue.

Advantageously, a preliminary histological, chemical or other study ofthe sample is carried out in order to define various areas of interestand to use them in the calculation of the TEC. Indeed, the TEC can belinked to a specific area of a sample, notably on a heterogeneoussample.

Generally, the TEC is obtained by the following relationship:

${T\; E\; C} = \frac{{Signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {target}\mspace{14mu} {analyte}\mspace{14mu} {on}\mspace{14mu} {the}\mspace{14mu} {support}}{{Signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {target}\mspace{14mu} {analyte}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {control}\mspace{14mu} {tissue}\mspace{14mu} {sample}}$

or inversely:

${T\; E\; C} = {\frac{{Signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {target}\mspace{14mu} {analyte}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {control}\mspace{14mu} {tissue}\mspace{14mu} {sample}}{{Signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {target}\mspace{14mu} {analyte}\mspace{14mu} {on}\mspace{14mu} {the}\mspace{14mu} {support}}.}$

The signal corresponds to the spectral characteristic of the massspectrum of the target analyte selected, for example the intensity ofthe peak of said analyte obtained on the mass spectrometer. Of course,the spectral characteristic used for the analyte in the reference mediumand on the sample must be the same.

The TEC is calculated using the same concentration of the target analytein a reference medium and on the sample which is to be analyzed. It isalso possible to use, in the place of the target analyte in a referencemedium, an analyte that has similar physicochemical properties to thoseof the target analyte (e.g. an isotopically labeled target analyte).

For the calculation of the TEC, the reference medium correspondsadvantageously to the sample support alone. To this end, for example,the analyte is solubilized in a suitable medium (organic solvent, wateror other) before deposition on the sample support. Deposition isfollowed by evaporation of the solvent, so that a dry deposition of theanalyte on said support is obtained. It is the signal obtained for saidanalyte on the sample support that is then used for the TEC.

The TEC value is generally the mean of several measurements of thetarget analyte under the same conditions, in order to obtain a reliablecoefficient.

Preferentially, the target analyte extinction coefficient (TEC) iscalculated only once for a given target analyte in a given type ofsample, and is reused for each analysis of said target analyte in thegiven type of sample. Thus, a database of TECs, listing the TEC of atarget analyte in several different samples, can advantageously becreated for a given target analyte, and used in each analysis of saidanalyte in various samples.

Alternatively, the TEC value can be determined prior to each analysis.

During step d), the weighted signal measured in the sample is used toquantify said analyte. Indeed, the value of the weighted signal onlydepends on the concentration of the analyte. It is thus possible, forexample, to determine the quantity of the target analyte by referring toa reference signal for the target analyte.

The expression “reference signal for the target analyte” refers to asignal representative of a known concentration, independent of thenature of the sample and its position in the sample. The referencesignal can be a mean or median value (or a range of mean or medianvalues) determined or established beforehand for a known quantity of agiven analyte. It can also be a standard curve.

According to the method selected and, if need be, to the referencesignal used, it is possible to determine the quantity of target analytein a relative or absolute manner.

For example, the reference signal is obtained by preparing a standardrange with at least three different known concentrations of the targetanalyte (or of another analyte with physicochemical properties similarto those of the target analyte), in a reference medium such as a samplesupport on which the solubilized analyte has been deposited. If theanalyte is deposited on a tissue sample, the analyte is advantageouslyadsorbed on said tissue after deposition and evaporation of thesolubilization medium.

An internal standard, different than the target analyte, can beintroduced into the standard range in order to normalize the signal ofsaid target analyte. The analyte used as internal standardadvantageously has physicochemical properties similar to those of thetarget analyte. A constant concentration of this standard is added tothe standard range of the target analyte before deposition.

Next, the mass spectrum for each concentration is analyzed. The spectralcharacteristic chosen as the comparison signal is read for each of saidconcentrations. Advantageously, after obtaining the mass spectra foreach concentration point, the selected associated spectralcharacteristic can be used to establish a calibration curve for saidanalyte. It is then sufficient to refer to this calibration curve todetermine precisely the concentration in the analyzed sample.

When the inventive method uses a mass spectrometry imaging techniquerequiring the use of a matrix, such as MALDI or matrix-enhancedsecondary ion mass spectrometry (ME-SIMS) imaging, it is possible to usea standard analyte to obtain the reference signal of the target analyte.

For example, a standard analyte, of known molecular weight and at aknown concentration, can be added to the MALDI matrix before use. Theresulting mixture is then deposited on the sample to be analyzed and onthe support before analysis step b). The signal obtained for thestandard analyte corresponds to the reference signal for the targetanalyte. By comparing the signal of the target analyte with thereference signal, the relative quantity of target analyte in the samplecan be deduced.

The standard analyte is any analyte whose molecular weight is known.Preferentially, an analyte with a molecular weight much different thanthe molecular weight of the target analyte is used as the standardanalyte, so that the mass spectra obtained can be easily analyzed. Theconcentration of the standard analyte, taken up in a solubilizationsolution (aqueous or containing a solvent), is defined in order not tosaturate the total signal.

The matrix/standard analyte mixture is deposited uniformly on the sampleas well as on the periphery of the sample, i.e., on the sample support,to enable calculation of the TEC. During drying, co-crystallization ofthe mixture can be observed with the naked eye.

The signal obtained for the standard analyte, whose concentration isknown, is used to normalize the spectral characteristics of the targetanalyte in order to enable its quantification. It is thus also possibleto take into account the effect of the matrix, described in furtherdetail below.

Another possibility for obtaining a reference signal (or internalstandard) is to use a deuterated analyte, i.e., an analyte labeled withdeuterium, or an analyte labeled with any other suitable isotope, as thestandard analyte.

For example, before analysis step b), a known concentration of thetarget analyte labeled with deuterium atoms can be added to the sampleto be analyzed. If a matrix is used, the deuterated analyte can be mixedwith the matrix. The resulting mixture is advantageously homogenizedbefore being deposited uniformly on the sample and the sample support.Otherwise, a solution containing the deuterated analyte can be depositedon the sample.

The target analyte and its deuterated complement can then be evaluatedsimultaneously on the analyzed sample. Considering that their ionizationwill be identical, their analysis by mass spectrometry will result inthe same signal with a difference in mass due to the presence ofdeuterium. The signal obtained for the deuterated analyte corresponds tothe reference signal for the target analyte. Since the concentration ofthe deuterated analyte is known, the ratio can then be calculated toyield a relative quantity.

The inventive method can advantageously be used with mass spectrometryimaging. In this case, it is possible to use various ionization sourcessuch as MALDI, laser desorption/ionization (LDI), desorptionelectrospray ionization (DESI), etc., combined with various types ofanalyzers such as time-of-flight (TOF), orbitrap, Fourier transform ioncyclotron resonance (FT-ICR), etc. This imaging technique makes itpossible to quantify the target analyte directly on the ion density mapobtained for the sample, corresponding to the spatial distribution ofthe target analyte in said sample. The weighted signal on said iondensity map can indeed be compared with a specific reference signal ofthe analyte of interest.

Certain mass spectrometry imaging techniques, such as MALDI or ME-SIMS,require the sample to be analyzed to be covered beforehand by a matrixcomprising small UV-absorbing organic molecules. This matrix enables thedesorption and the ionization of the molecules present on the sample.

The inventive method can be used regardless of the matrix chosen. Thesematrices are provided in solid form (crystallization on the sample) orliquid form and are ionic or non-ionic. The matrix is chosen accordingto the mass range analyzed. They are generally prepared immediatelybefore use in a solvent/aqueous solution mixture.

Several methods for depositing the matrix are possible, notably manualdeposition using a pipette, which makes it possible to deposit a precisevolume of matrix directly on the sample. It is also possible to depositthe matrix by spraying or by nebulization, wherein the matrix is sprayedor nebulized directly on the tissue by a robotic system or manually.Similarly, deposition by microdroplets wherein the matrix is spotted onthe sample via piezoelectric, acoustic or syringe pump systems can beenvisaged. It is also possible to deposit the matrix by sifting, inorder to deposit the matrix in solid form.

Advantageously, if the inventive method uses MALDI mass spectrometryimaging, a step of evaluating the homogeneity of the deposition ofmatrix on the sample can be expected. Indeed, the signal correspondingto the matrix used can indicate the quality/uniformity of the depositionof said matrix. Matrix defects on the surface of the sample can then becorrelated with the lack of detection or the loss of intensity of thesignal of the target analyte in the sample studied.

The homogeneity of the matrix can be evaluated according to qualitativecriteria by observing under an optical microscope the homogeneity of thedeposition on the surface of the sample, and/or according toquantitative criteria by monitoring variations in the signal relative tothe matrix itself on the sample.

With regard to qualitative criteria, it must be ensured that the matrixhas been deposited as uniformly as possible on the surface under studyand that there are no areas void of matrix and that its crystallizationis optimal.

For quantitative evaluation of the homogeneity of the matrix deposition,the matrix is considered as an analyte itself whose signal during sampleanalysis is detected in the same way as the signal of the targetanalyte. The signal of the matrix molecule is then compared with itsreference signal. The reference signal of the matrix corresponds in thiscase to the signal emitted by the matrix on a deposition of referencematrix, i.e., on a sample and on a sample support used specifically tomeasure the reference signal of the matrix.

By these additional steps, the signal of the target analyte is validatedand normalized to take into account variation in the quality of thematrix deposition, which can affect the matrix deposition's spectralcharacteristics.

This consideration of the matrix effect can be particularly advantageouswhen the monitoring of changes in the presence of a target analyte overtime is desired, since matrix deposition quality can vary from onesample to the next.

The inventive method can be used to analyze any kind of molecule (targetanalyte), such as, for example, peptides, polypeptides, proteins, aminoacids, nucleic acids, lipids, metabolites, etc., and, in general, anyanalyte that is active pharmaceutically or otherwise and that can beionized by mass spectrometry. The inventive method is particularlyadvantageous for the analysis of small analytes (notably drugs), i.e.,analytes of molecular weight less than 2000 Da.

If the target analyte is a protein of high molecular weight, it ispossible to enzymatically and/or chemically pretreat the target analytein order to cleave it into several peptides (FIG. 2). Detection andquantification are then carried out for at least one of the peptidesresulting from the enzymatic digestion and/or the chemicaldegradation/modification, representative of said protein. For example,trypsin can be used as an enzyme to cleave the target protein intoseveral peptides identified beforehand. Chemical pretreatment canconsist of chemical hydrolysis, by acids or bases, a Maillard reaction,the formation of isopeptides or lysinoalanine, etc.

Similarly, it is possible to treat the sample to be analyzed with atleast one solvent and/or at least one detergent prior to detection stepb) so as to optimize detection of the target analyte. For example,washing with chloroform (FIG. 1-a) removes certain classes of lipids.Washing with ethanol (FIG. 1-b) enables better detection of low-massanalytes. These two washings, in the experiment illustrated in FIG. 1,remove certain analytes, in particular lipids, thus promoting thedetection of new ions directly on the tissue.

The inventive method can also be used to detect and quantify at leasttwo different target analytes on the same sample, simultaneously orsequentially.

The inventive method is particularly suited to the detection andquantification of target analytes on a section of biological, plant oranimal tissue. Notably, analysis on a section of whole animal can makeit possible to compare, on the same sample, the distribution of targetanalytes in various tissues of said animal.

According to the invention, the source data, i.e., the TEC of the targetanalyte and the matrix effect, can be normalized with a view toquantification by means of a computer program that integrates all orpart of these factors. This computer program, or data analysis software,advantageously uses the TEC value weighted, if need be, by the matrixeffect during the processing of the image in the case of massspectrometry imaging.

Another object of the invention thus relates to a computer-readable datamedium comprising computer-executable instructions, such as, forexample, the reading of raw data resulting from mass spectrometryanalysis, and/or determination of TECs and/or determination of thecalibration curve, and/or normalization of the raw data using said TECsor said calibration curve in order to obtain a quantitative value forthe target analyte. Advantageously, these computer-executableinstructions are suited to enable a computer system to execute at leaststep c) of the inventive method.

The data medium advantageously comprises at least one TEC database forat least one target analyte in at least two different sample types.Preferentially, in the case of biological samples, such as a section ofwhole animal, the database lists the TECs of at least one target analytein various tissues of said sample.

The data medium can also comprise a database of the reference signal ofat least one matrix used in mass spectrometry imaging. Thus, by using animaging method that makes use of the data medium, it is possible to takeinto account the matrix effect during the analysis of the sample.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Examples of the effect of two washings in direct analysisby mass spectrometry of heart tissue. (a): Signal obtained from hearttissue without any preliminary washing of the matrix deposition. (b):Signal obtained from heart tissue on a section adjacent to (a) using a90% ethanol washing prior to deposition of the matrix. (c): Signalobtained from stomach tissue on a section adjacent to (a) using a 100%chloroform washing prior to deposition of the matrix.

FIGS. 2A-2B. MALDI-TOF mass spectrum of a digested model protein (bovineserum albumin with trypsin as digestion enzyme) on (a) a referencesupport (slide) and (b) tissue (rat liver). Example of the m/z 928fragment (YLYEIAR): observation of the extinction of the mass signalsrelative to the peptide on the tissue, with the TEC equal to 1.62.

FIG. 3. A schematic representation of the principal steps of theinventive method, according to an example of implementation using massspectrometry imaging.

FIGS. 4A-4B. Methodology for calculating the tissue extinctioncoefficient in the study of the distribution of propranolol inwhole-body mouse. (a) Optical image of a sagittal section of controlmouse for visualizing various organs or target areas (1—slide alone,2—brain, 3—kidney, 4—lung, 5—liver, 6—heart). (b) Mass spectrometryimage of the distribution of the internal standard (propranolol, [M+H]⁺ion at m/z 260) mixed with the matrix on and apart from the sample andintensity scale.

FIGS. 5A-5B. Methodology for calculating the tissue extinctioncoefficient of propranolol in the kidney. FIG. 5A: Optical image of thekidney, delimitation of several regions of interest (ROI) or areas ofpoints of equal dimensions within the target organ. FIG. 5B: Table ofmean intensities of the internal standard by ROI and between ROI (ROImean). FIGS. 6A-6B. FIG. 6A: Table summarizing propranolol intensitiesby organ or by target areas. FIG. 6B: Histogram of propranololintensities by organ or by target areas.

FIGS. 7A-7B. Calculation of the tissue extinction coefficient. FIG. 7A:Mathematical relationship: Table summarizing TECs calculated forpropranolol by target organ. FIG. 7B: Histogram of TECs calculated forpropranolol by target organ.

FIGS. 8A-8C: Determination of the calibration curve for the targetanalyte. FIG. 8A: Optical image of standard range depositions. FIG. 8B:Mass spectrometry image of the standard range, distribution of thetarget analyte. FIG. 8C: Table summarizing mean intensities of ROI ofthe standard range of the target analyte.

FIGS. 9A-9C. FIG. 9A: Table summarizing mean intensities of ROI of thestandard range of the target analyte. FIG. 9B: Graph of the calibrationline. FIG. 9C: Correlation coefficient and straight-line equation.

FIGS. 10A-10C. Quantification on the mass spectrometry image of thetarget analyte (propranolol), in a whole body. FIG. 10A: Optical imageof a sagittal section of mice at 20 min post—injection of the targetanalyte and visualization of various organs or target areas (2—brain,3—kidney, 4—lung, 5—liver, 6—heart). FIG. 10B: Mass spectrometry imageof the distribution at t=60 min post-injection of the target analyte([M+H]⁺ ion at m/z 260) by intensity. FIG. 10C: Mass spectrometry imageof the distribution at t=20 min post-injection of the target analyteafter normalization by the TEC and correlation with the calibrationline, access to the quantity per unit of area of the target analyte.

FIG. 11. Quantification on the MS image of the target analyte, tablesummarizing the quantity of target analyte in the various organs.Explanation of the methodology for calculating the latter from meanintensities by organ and pixel with use of the TEC.

FIGS. 12A-12C. Methodology for calculating the tissue extinctioncoefficient of olanzapine in the kidney. FIG. 12A: Optical image of asagittal section of control mouse kidney. FIG. 12B: Mass spectrometryimage of the distribution of the internal standard (olanzapine, [M+H]⁺ion at m/z 313.3) mixed with the matrix on and apart from the sample andintensity scale. FIG. 12C: Histogram of the TEC calculated forolanzapine in the kidney.

FIGS. 13A-13B. Determination of the calibration curve for the targetanalyte. FIG. 13A: Mass spectrometry image of the standard range,distribution of the target analyte. FIG. 13B: Presentation of thecalibration line relative to the MS image of olanzapine, its equation,its correlation coefficient and its limits of detection and ofquantification in fmol/mm².

FIGS. 14A-14B. Quantification using the mass spectrometry image of thetarget analyte (olanzapine), in a kidney treated for 2 hours. FIG. 14A:Optical image of a sagittal section of mouse kidney at 2 hourspost-administration of the target analyte. FIG. 14B: Mass spectrometryimage of the distribution at t=2 h post-administration of the targetanalyte ([M+H]⁺ ion at m/z 313.3) by intensity.

EXAMPLES

The inventive method will now be described in further detail usingspecific examples and the figures presented above. These examples aregiven for illustrative purposes only and by no means restrict the scopeof the invention.

Example 1

In example 1, the distribution of a drug (propranolol) in various organsof a mouse is studied by MALDI mass spectrometry imaging. Of course, inan almost identical manner an imaging device other that MALDI could beused, such as, for example, the following sources: SIMS, DESI, DIOS,ICP, MALDI microscope, SNOM, SMALDI, LA-ICP, ESI (liquid extraction ontissue), MILDI, JEDI, ELDI, etc.

Materials and Methods

Materials:

-   -   2,5-Dihydroxybenzoic acid (DHB) (Sigma-Aldrich, Saint-Quentin        Fallavier, France)    -   Trifluoroacetic acid (TFA) (Sigma-Aldrich)    -   Methanol (Sigma-Aldrich)    -   Propranolol (Sigma-Aldrich)

Animals:

Male Swiss mice weighing 25-40 g (Charles River, France) were used.Propranolol taken up in 0.9% NaCl solution was injected by intravenousroute at a concentration of 7.5 mg/kg.

The animals were sacrificed by CO₂ asphyxiation at 20 minutespost-injection.

The animals were then plunged into 100% isopentane solution cooled byliquid nitrogen for rapid freezing.

The animals were then stored at −80° C.

Preparation of Samples for Mass Spectrometry:

The samples (control and dosed tissue) were sectioned into 20 μm-thicklayers using a CM1510S cryostat (Leica Microsystems, Nanterre, France)cooled at −26° C. The sections were then deposited on conductive ITO(indium tin oxide) slides (Bruker Daltonics, Bremen, Germany).

Finally, the sections were placed in a desiccator for 20 minutes.

Preparation for Acquisition by MALDI Imaging:

A DHB matrix was used for the analysis of the target analyte(propranolol) in the dosed tissue sections. This matrix was prepared ata concentration of 40 mg/ml in methanol/0.1% TFA (1:1, v/v). The matrixsolution was deposited using the SunCollect spraying system (SunChrome,Germany).

On the same slide but apart from the dosed tissue section, a range ofdilutions of propranolol taken up in water was deposited manually usinga pipette (1 μl per point) prior to deposition of the matrix. This rangeof dilutions extends from 10 pmol/μl to 0.02 pmol/μl and includes sevenpoints.

Preparation for Calculation of the TEC:

On a control tissue section, a 10 mg/ml HCCA matrix solution in ACN/0.1%TFA (7:3, v/v) was also prepared, to which a 10 pmol/μl propranololsolution was added. The matrix solution was deposited using theSunCollect spraying system (SunChrome, Germany) to cover the surface ofthe control tissue section.

MALDI Image Acquisition:

The images were obtained using an AutoFlex Speed MALDI-TOF massspectrometer (Bruker Daltonics, Bremen, Germany) equipped with aSmartbeam laser. The data was generated in positive reflectron mode. Atotal of 700 spectra were obtained for each spot with a 1000 Hz laserfrequency and a 300×300 μm² image spatial resolution on a mass range of100 Da to 1000 Da. The Flexlmaging version 2.1 software was used toreconstruct the images.

1—Calculation of the TEC of the Target Analyte (Propranolol) on aControl Sample

A sagittal section of the whole animal, prepared as specified above andused as a control sample, is deposited on a slide.

The propranolol solution used as an internal standard is mixed with thematrix above, prior to deposition of the resulting mixture on thecontrol sample.

The control sample is then analyzed by mass spectrometry imaging inorder to obtain an image of the distribution of the internal standard onthe control sample and on the support slide (FIG. 3B).

The various organs of interest can advantageously be located beforehandby optical imaging of the control sample (FIG. 3A).

Several regions of interest (ROI) of equal dimensions for each organ onthe slide are then delimited on the mass spectrometry image of thecontrol sample. The intensity of the peaks of the mass spectrum wasselected as the reference signal. The mean intensities of the internalstandard for each ROI and each organ of interest were recorded. For eachorgan, a mean intensity (ROI mean) was calculated from the intensityobtained for all the corresponding ROI. This mean ROI will be used tocalculate the extinction coefficient of propranolol in each organstudied.

FIG. 5 shows a schematic diagram of these various steps for the kidney.

FIG. 6A summarizes the mean ROI obtained for propranolol in the variousorgans of interest of the control sample, and FIG. 6B shows thehistogram of the corresponding signal intensities obtained.

The TEC can then be calculated (FIG. 7A) using the mean ROI values fromthe slide and from each organ, according to the mathematical formula:

${T\; E\; C} = \frac{{{Int}({slide})}_{x}}{{{Int}({tissue})}_{x}}$

Thus, for the same concentration of propranolol, the associated signalin the kidney is divided by nearly 13 compared to the expected signal,i.e., the signal on the slide. On the other hand, the signal is onlydivided by 4.82 in the liver and 7.96 in the heart. The signal isdivided by less than 8 in the lungs and by 6.18 in the brain.

2—Propranolol Calibration Curve

In the example described herein using FIGS. 8 and 9, the referencesignal for the target analyte (propranolol) corresponds to the signalobtained for a standard range of seven concentrations of propranolol.

Seven droplets of a propranolol and matrix solution, corresponding toseven different concentrations of propranolol (0 to 3 pmol/μl), aremanually deposited on a slide using a pipette and allowed to dry. Tomake it easier to read the results, the droplets are deposited inincreasing concentrations and sufficiently spaced to avoid any risk ofoverlapping.

The mass spectrometry image obtained for these various concentrations(FIG. 8B) is normalized for all points in the range using an ROI ofidentical dimensions, from which a mean reference intensity, orreference signal, for propranolol is defined. A calibration line (FIG.9B) can then be plotted, thereafter making it possible during analysisto correlate any signal intensity obtained for propranolol with aconcentration by pixel.

3—Quantification of Propranolol Directly on the Mass Spectrometry Imageof the Sample

A sagittal section of the whole animal, prepared as specified above andif possible in the same plane as the section used as the control sampleduring the calculation of the TEC, is deposited on a slide in order toperform an analysis by mass spectrometry imaging (FIG. 10B).

The signal intensity obtained for propranolol is increased in each organof interest as a function of the TEC calculated for each.

A mass spectrometry image of the section of the whole animal is obtainedin which signal intensity corresponds to absolute intensity, i.e.,intensity of the propranolol concentration alone (FIG. 10C). This imagecan be correlated with the calibration line previously calculated forpropranolol in order to determine the quantity of propranolol in thesample directly by visualizing the image.

Thus, in the example described herein, it is noted that propranolol isvirtually absent from the liver and lungs, in contrast to the brainwhere the distribution of propranolol is greatest with a total quantityof the target analyte of about 5 ng. Propranolol is also observed in thekidneys and lungs, with quantities ranging from 1.28 ng to 2 ng.

Example 2

In the second example, the distribution of another drug (olanzapine) inthe kidneys of a mouse is studied by MALDI mass spectrometry imaging.Any other imaging device could be used in a virtually identical manner.

Materials and Methods

Materials:

-   -   Hydroxycinnamic acid (HCCA) (Sigma-Aldrich, Saint-Quentin        Fallavier, France),    -   Trifluoroacetic acid (TFA) (Sigma-Aldrich)    -   Acetonitrile, DMSO, water (Sigma-Aldrich)    -   Olanzapine (Lilly Research Laboratories, Eli Lilly and Co.,        Indianapolis, Ind.)

Animals:

Male Swiss mice weighing 25-40 g (Charles River, France) were used.Olanzapine was administered orally at a concentration of 8 mg/kg.

The animals were sacrificed by CO₂ asphyxiation at 2 hourspost-administration.

The animals were then plunged into 100% isopentane solution cooled byliquid nitrogen for rapid freezing.

The animals were then stored at −80° C.

Preparation of Samples for Mass Spectrometry:

The samples were sectioned into 10 μm-thick layers under conditionsidentical to those of example 1 above.

Preparation for Acquisition by MALDI Imaging:

An HCCA matrix was used for the analysis of the target analyte(olanzapine) in the tissue sections. This matrix was prepared at aconcentration of 10 mg/ml in ACN/0.1% TFA (7:3, v/v). The matrixsolution was deposited using the SunCollect spraying system.

On the same slide but apart from the tissue section, a range ofdilutions of olanzapine taken up in DMSO was deposited manually using apipette (1 μl per point) prior to deposition of the matrix. This rangeof dilution extends from 60 pmol/μl to 1 pmol/μl and includes sevenpoints.

Preparation for Calculation of the TEC:

On a control tissue section adjacent to that used for MALDI imageacquisition, a 10 mg/ml HCCA matrix solution in ACN/0.1% TFA (7:3, v/v)was also prepared, to which a 10 pmol/μl olanzapine solution was added.The matrix solution was deposited using the SunCollect spraying systemto cover the surface of the tissue section.

MALDI Image Acquisition:

The images were obtained in a manner identical to example 1, but with a200×200 μm² image spatial resolution.

1—Calculation of the TEC of the Target Analyte (Olanzapine) on a ControlSample

A control sagittal section of kidney, prepared as specified above andused as a control sample, is deposited on a slide. The olanzapinesolution used as an internal standard is mixed with the matrix above,prior to deposition of the resulting mixture on the control sample.

The control sample is then analyzed by mass spectrometry imaging inorder to obtain an image of the distribution of the internal standard onthe control sample and on the support slide (FIG. 12B). The opticalimage of the control sample presented in FIG. 12A makes it possible tovisualize the organ of interest, i.e., the kidney.

Several regions of interest (ROI) of equal dimensions on the kidney andon the slide are then delimited on the mass spectrometry image of thecontrol sample. The intensity of the peaks of the mass spectrum wasselected as the reference signal. The mean intensity (ROI mean) iscalculated as for example 1. This mean ROI will be used to calculate theextinction coefficient of olanzapine in the kidney.

The TEC (FIG. 12C) is calculated according to same methodology asexample 1. It is thus observed that, for the same concentration ofolanzapine, the associated signal in the kidney is divided by nearly22.2 compared to the expected signal, i.e., the signal on the slide.

2—Olanzapine Calibration Curve

The reference signal for olanzapine, represented in FIG. 13, correspondsto the signal obtained for a standard range of seven concentrations ofolanzapine.

Seven droplets of a olanzapine and matrix solution, corresponding toseven different concentrations of olanzapine (0 to 60 pmol/μl), aremanually deposited on a slide using a pipette and allowed to dry. Tomake it easier to read the results, the droplets are deposited inincreasing concentrations and sufficiently spaced to avoid any risk ofoverlapping.

The mass spectrometry image obtained for these various concentrations(FIG. 13A) is normalized for all points in the range using an ROI ofidentical dimensions, from which a mean reference intensity, orreference signal, for olanzapine is defined. A calibration line (FIG.13B) can then be plotted, thereafter making it possible during analysisto correlate any signal intensity obtained for olanzapine with aconcentration in pmol per mm².

3—Quantification of Olanzapine Using the Mass Spectrometry Image of theSample

A sagittal section of kidney treated with olanzapine, prepared asspecified above and if possible in the same plane as the section used asthe control sample during the calculation of the TEC, is deposited on aslide in order to perform an analysis by mass spectrometry imaging (FIG.14A). A mass spectrometry image of the section of kidney is thusobtained, on which olanzapine is localized through the detection of them/z 313.3 ion (FIG. 14B) primarily in the medulla.

The signal intensity obtained for olanzapine is normalized in the kidneyas a function of the TEC calculated beforehand. This data can then becorrelated with the calibration line previously calculated forolanzapine in order to obtain its total concentration in the kidney inpmol/mm².

With knowledge of the thickness and the surface area of the kidneysection analyzed it is then possible to determine the quantity ofolanzapine in grams per gram of tissue in the sample.

Thus, in the example described herein, a mean concentration ofolanzapine (over three experiments) of 41.1 μg/g tissue can becalculated.

We claim:
 1. A method for detecting and quantifying by mass spectrometryimaging at least one target analyte in a sample, comprising thefollowing steps: a) depositing the sample containing at least one targetanalyte to be analyzed on a support for mass spectrometry imaging anddepositing a known concentration of isotopically labeled target analyte,as an internal standard, on said sample; b) analyzing the sample by amass spectrometry imaging technique so as to obtain a mass spectrumsignal of said at least one target analyte and a mass spectrum signal ofthe isotopically labeled target analyte in said sample; c) weighting thesignal associated with the mass spectrum of said at least one targetanalyte in said sample by an extinction coefficient specific to each ofsaid at least one target analyte and to the sample, wherein saidextinction coefficient corresponds to a ratio of the signal of the atleast one target analyte to the signal of the isotopically labeledtarget analyte, or the inverse; and d) using each weighted signal ofeach of said at least one target analyte to determine the quantity anddistribution of each of said at least one target analyte in the sample.2. The method according to claim 1, wherein said at least one targetanalyte is a protein, a peptide, a lipid, a metabolite or a smallmolecule.
 3. The method according to claim 1, wherein the sample is anorganic sample or an inorganic sample.
 4. The method according to claim1, wherein the sample is a biological sample.
 5. The method according toclaim 1, wherein said at least one target analyte is a protein and anenzymatic and/or chemical pretreatment of said protein is performedprior to detection step b), and wherein detection and quantification arecarried out for at least one of the peptides resulting from saidpretreatment.
 6. The method according to claim 1, wherein the sample istreated with at least one solvent and/or at least one detergent prior toanalysis step b).
 7. The method according to claim 1, wherein the massspectrum signal corresponds to the intensity of the peak, the area ofthe peak or the signal-to-noise ratio of the mass spectrum of at leastone target analyte.
 8. The method according to claim 1, wherein thesignal intensity of the target analyte is weighted directly on theanalyzed sample so as to simultaneously visualize the distribution andthe concentration of the target analyte in said sample.
 9. The methodaccording to claim 1, wherein the isotopically labeled target analyte islabeled with deuterium atoms.
 10. The method according to claim 1,wherein a known concentration of the isotopically labeled target analyteis mixed with a matrix before spreading the resulting mixture over thetissue sample.
 11. The method according to claim 1, wherein theisotopically labeled target analyte is solubilized in a solvent beforedeposition on the sample and the support, and an evaporation of thesolvent is performed before the mass spectrometry imaging analysis. 12.The method according to claim 1, wherein a matrix is deposited on thesample before analysis step b) and wherein before analysis step b) thehomogeneity of the matrix deposition on the sample is evaluated inrelation to a deposition of a reference matrix.
 13. The method accordingto claim 1, wherein at least two different target analytes in saidsample are detected and quantified simultaneously.
 14. The methodaccording to claim 1, wherein the sample is a section of whole animaland wherein the target analyte is detected and quantified by massspectrometry imaging directly on said section so as to simultaneouslycompare the distribution of said target analyte in various tissues ofthe animal.
 15. A computer-readable data medium comprisingcomputer-executable instructions suited to enable a computer system toexecute step c) of the method according to claim
 1. 16. The methodaccording to claim 1, wherein the target analyte is ionizable.